The goals of the proposed work are twofold. The initial goal is to develop chemical and physical techniques for the detection of motions of the polypeptide backbone in proteins, including movements of entire domains. These techniques will be used to develop a molecular picture of backbone and domain motions in a specific protein, the galactose binding protein of E. coli. Simultaneously the biological role of motions in the sensory and transport functions of this protein will be addressed. The long term goal is to contribute to an understanding of the general physical principles underlying large-scale motions in proteins, and to ascertain the biological roles of these motions. To address the first goal, the structure of the galactose protein, known to 1.9 angstrom resolution, will be examined for motions as follows. 1) Site-directed mutagenesis will be used to place a pair of cysteine residues at chosen locations within the average structure of the protein. 2) Internal motions which yield collision of the mutant cysteines will be detected by disulfide bond formation between mutant cysteines. If the alpha-carbons of the cysteines are greater than 7 angstrom apart in the average structure, then a motion detected by disulfide formation must involve a change in the backbone structure. 3) The outlined approach will be systemically repeated for different pairs of mutant cysteine locations. This will enable the ranges of motion of alpha-helices, beta-sheets, loops, and free termini within a given domain, as well as the movements of entire domains, to be mapped out. 4) The structural basis of the detected motions, which are effectively trapped by disulfide formation, will be analyzed by optical, NMR and X-ray crystallographic techniques. 5) The timescales of the detected motions will be examined by measurements of disulfide formation rates for slow motions, and by spectroscopic analysis of motional probes covalently attached to mutant cysteines for more rapid motions. 6) The biological role of the detected motions in the sensory and transport functions of the protein will be investigated by a) measuring the effect of substrates and cofactors on motions, and b) using disulfide bond formation to lock the protein in active and inactive conformers. The structures of trapped conformers will be ascertained by optical, NMR and X-ray crystallographic techniques. To address the second goal, the backbone and domain motions of proteins in general, as well as the structural factors which modulate these motions, will ultimately be investigated by applying to other proteins the chemical and physical approaches developed for the galactose binding protein. A potential application of this technology is the use of disulfide bond formation to activate or inactivate enzymes and signalling proteins, in a covalent yet reversible manner. In addition, bacterial binding proteins have been useful as detectors in assays for genetic diseases and treatment (reviewed in ref. 39).